Heat Shock Sensitivity Research Articles

Hyperthermia Selectively Destabilizes Oncogenic Fusion Proteins.

Hyperthermia destabilizes oncofusion proteins including PML/RARα and acts synergistically with standard arsenic therapy in relapsed and refractory APL. The results open up the possibility that heat shock sensitivity may be an easily targetable vulnerability of oncofusion-driven cancers.See related commentary by Wu et al., p. 300.

Multiplexed mRNA assembly into ribonucleoprotein particles plays an operon-like role in the control of yeast cell physiology.

Prokaryotes utilize polycistronic messages (operons) to co-translate proteins involved in the same biological processes. Whether eukaryotes achieve similar regulation by selectively assembling and translating monocistronic messages derived from different chromosomes is unknown. We employed transcript-specific RNA pulldowns and RNA-seq/RT-PCR to identify yeast mRNAs that co-precipitate as ribonucleoprotein (RNP) complexes. Consistent with the hypothesis of eukaryotic RNA operons, mRNAs encoding components of the mating pathway, heat shock proteins, and mitochondrial outer membrane proteins multiplex in trans, forming discrete messenger ribonucleoprotein (mRNP) complexes (called transperons). Chromatin capture and allele tagging experiments reveal that genes encoding multiplexed mRNAs physically interact; thus, RNA assembly may result from co-regulated gene expression. Transperon assembly and function depends upon histone H4, and its depletion leads to defects in RNA multiplexing, decreased pheromone responsiveness and mating, and increased heat shock sensitivity. We propose that intergenic associations and non-canonical histone H4 functions contribute to transperon formation in eukaryotic cells and regulate cell physiology.

The sensitivity of the yeast, Saccharomyces cerevisiae, to acetic acid is influenced by DOM34 and RPL36A.

The presence of acetic acid during industrial alcohol fermentation reduces the yield of fermentation by imposing additional stress on the yeast cells. The biology of cellular responses to stress has been a subject of vigorous investigations. Although much has been learned, details of some of these responses remain poorly understood. Members of heat shock chaperone HSP proteins have been linked to acetic acid and heat shock stress responses in yeast. Both acetic acid and heat shock have been identified to trigger different cellular responses including reduction of global protein synthesis and induction of programmed cell death. Yeast HSC82 and HSP82 code for two important heat shock proteins that together account for 1–2% of total cellular proteins. Both proteins have been linked to responses to acetic acid and heat shock. In contrast to the overall rate of protein synthesis which is reduced, the expression of HSC82 and HSP82 is induced in response to acetic acid stress. In the current study we identified two yeast genes DOM34 and RPL36A that are linked to acetic acid and heat shock sensitivity. We investigated the influence of these genes on the expression of HSP proteins. Our observations suggest that Dom34 and RPL36A influence translation in a CAP-independent manner.

Loss-of-Function Mutations in HspR Rescue the Growth Defect of a Mycobacterium tuberculosis Proteasome Accessory Factor E (pafE) Mutant.

Mycobacterium tuberculosis uses a proteasome to degrade proteins by both ATP-dependent and -independent pathways. While much has been learned about ATP-dependent degradation, relatively little is understood about the ATP-independent pathway, which is controlled by Mycobacterium tuberculosisproteasome accessory factor E (PafE). Recently, we found that a Mycobacterium tuberculosispafE mutant has slowed growth in vitro and is sensitive to killing by heat stress. However, we did not know if these phenotypes were caused by an inability to degrade the PafE-proteasome substrate HspR (heat shock protein repressor), an inability to degrade any damaged or misfolded proteins, or a defect in another protein quality control pathway. To address this question, we characterized pafE suppressor mutants that grew similarly to pafE+ bacteria under normal culture conditions. All but one suppressor mutant analyzed contained mutations that inactivated HspR function, demonstrating that the slowed growth and heat shock sensitivity of a pafE mutant were caused primarily by the inability of the proteasome to degrade HspR.IMPORTANCEMycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required for virulence. We recently discovered a proteasome cofactor, PafE, which is required for the normal growth, heat shock resistance, and full virulence of M. tuberculosis In this study, we demonstrate that PafE influences this phenotype primarily by promoting the expression of protein chaperone genes that are necessary for surviving proteotoxic stress.

Molecular mechanisms of nutlin-3 involve acetylation of p53, histones and heat shock proteins in acute myeloid leukemia.

BackgroundThe small-molecule MDM2 antagonist nutlin-3 has proved to be an effective p53 activating therapeutic compound in several preclinical cancer models, including acute myeloid leukemia (AML). We and others have previously reported a vigorous acetylation of the p53 protein by nutlin-treatment. In this study we aimed to investigate the functional role of this p53 acetylation in nutlin-sensitivity, and further to explore if nutlin-induced protein acetylation in general could indicate novel targets for the enhancement of nutlin-based therapy.ResultsNutlin-3 was found to enhance the acetylation of p53 in the human AML cell line MOLM-13 (wild type TP53) and in TP53 null cells transfected with wild type p53 cDNA. Stable isotope labeling with amino acids in cell culture (SILAC) in combination with immunoprecipitation using an anti-acetyl-lysine antibody and mass spectrometry analysis identified increased levels of acetylated Histone H2B, Hsp27 and Hsp90 in MOLM-13 cells after nutlin-treatment, accompanied by downregulation of total levels of Hsp27 and Hsp90. Intracellular levels of heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70 and Hsp90α were correlated to nutlin-sensitivity for primary AML cells (n = 40), and AML patient samples with low sensitivity to nutlin-3 tended to express higher levels of heat shock proteins than more responsive samples. Combination therapy of nutlin-3 and Hsp90 inhibitor geldanamycin demonstrated synergistic induction of apoptosis in AML cell lines and primary AML cells. Finally, TP53 null cells transfected with a p53 acetylation defective mutant demonstrated decreased heat shock protein acetylation and sensitivity to nutlin-3 compared to wild type p53 expressing cells.ConclusionsAltogether, our results demonstrate that nutlin-3 induces acetylation of p53, histones and heat shock proteins, and indicate that p53 acetylation status and the levels of heat shock proteins may participate in modulation of nutlin-3 sensitivity in AML.

P120 Catenin Is Required for the Stress Response in Drosophila

p120ctn is a ubiquitously expressed core component of cadherin junctions and essential for vertebrate development. Surprisingly, Drosophila p120ctn (dp120ctn) is dispensable for adherens junctions and development, which has discouraged Drosophila researchers from further pursuing the biological role of dp120ctn. Here we demonstrate that dp120ctn loss results in increased heat shock sensitivity and reduced animal lifespan, which are completely rescued by ectopic expression of a dp120ctn-GFP transgene. Transcriptomic analysis revealed multiple relish/NF-κB target genes differentially expressed upon loss of dp120ctn. Importantly, this aberrant gene expression was rescued by overexpression of dp120ctn-GFP or heterozygosity for relish. Our results uncover a novel role for dp120ctn in the regulation of animal stress response and immune signalling. This may represent an ancient role of p120ctn and can influence further studies in Drosophila and mammals.

Ras Protein/cAMP-dependent Protein Kinase Signaling Is Negatively Regulated by a Deubiquitinating Enzyme, Ubp3, in Yeast

Ras proteins and cAMP-dependent protein kinase (protein kinase A, PKA) are important components of a nutrient signaling pathway that mediates cellular responses to glucose in yeast. The molecular mechanisms that regulate Ras/PKA-mediated signaling remain to be fully understood. Here, we provide evidence that Ras/PKA signaling is negatively regulated by a deubiquitinating enzyme, Ubp3. Disrupting the activity of Ubp3 leads to hyperactivation of PKA, as evidenced by much enhanced phosphorylation of PKA substrates, decreased accumulation of glycogen, larger cell size, and increased sensitivity to heat shock. Levels of intracellular cAMP and the active forms of Ras proteins are also elevated in the ubp3Δ mutant. Consistent with a possibility that the increased cAMP is responsible for the abnormal signaling behavior of the ubp3Δ mutant, overexpressing PDE2, which encodes a phosphodiesterase that hydrolyzes cAMP, significantly relieves the cell size increase and heat shock sensitivity of the mutant. Further analysis reveals that Ubp3 interacts with a Ras GTPase-accelerating protein, Ira2, and regulates its level of ubiquitination. Together, our data indicate that Ubp3 is a new regulator of the Ras/PKA signaling pathway and suggest that Ubp3 regulates this pathway by controlling the ubiquitination of Ras GTPase-accelerating protein Ira2.

Hyperthermia induces cytoskeletal alterations and mitotic catastrophe in p53-deficient H1299 lung cancer cells

Hyperthermia is used in cancer therapy, however much remains to be discovered regarding its mechanisms of action at the cellular level. In this study, the effects of hyperthermia on cell death, survival, morphology and the cytoskeleton were investigated in a non-small cell lung cancer cell line, H1299. Despite the fact that this cell line is widely used in research, it has not yet been tested for heat shock sensitivity. Cells were given a 30-min heat shock at 43.5°C and 45°C and left to recover at 37°C for 24 and 48h. 24h after heat shock treatment, we monitored changes in the organization of the cytoskeleton using immunofluorescence microscopy. The number of actin stress fibers was significantly reduced, microtubules formed a looser meshwork, a portion of the cells possessed multipolar mitotic spindles, whereas vimentin filaments collapsed into perinuclear complexes. 48h following heat stress, most of the cells showed recovery of the cytoskeleton, however we observed a considerable number of giant cells that were multinucleated or contained one enlarged nucleus. The data obtained by MTT assay showed a dose-dependent decrease of cell viability, while flow cytometric analysis revealed an increase in the number of cells with externalized phosphatidylserine. The results suggest that one of the modes of heat-induced cell death in H1299 cells is mitotic catastrophe, which probably ends in apoptosis.

VapC6, a ribonucleolytic toxin regulates thermophilicity in the crenarchaeote Sulfolobus solfataricus

The phylum Crenarchaeota includes hyperthermophilic micro-organisms subjected to dynamic thermal conditions. Previous transcriptomic studies of Sulfolobus solfataricus identified vapBC6 as a heat-shock (HS)-inducible member of the Vap toxin-antitoxin gene family. In this study, the inactivation of the vapBC6 operon by targeted gene disruption produced two recessive phenotypes related to fitness, HS sensitivity and a heat-dependent reduction in the rate of growth. In-frame vapBC6 deletion mutants were analyzed to examine the respective roles of each protein. Since vapB6 transcript abundance was elevated in the vapC6 deletion, the VapC6 toxin appears to regulate abundance of its cognate antitoxin. In contrast, vapC6 transcript abundance was reduced in the vapB6 deletion. A putative intergenic terminator may underlie these observations by coordinating vapBC6 expression. As predicted by structural modeling, recombinant VapC6 produced using chaperone cosynthesis exhibited heat-dependent ribonucleolytic activity toward S. solfataricus total RNA. This activity could be blocked by addition of preheated recombinant VapB6. In vivo transcript targets were identified by assessing the relative expression of genes that naturally respond to thermal stress in VapBC6-deficient cells. Preferential increases were observed for dppB-1 and tetR, and preferential decreases were observed for rpoD and eIF2 gamma. Specific VapC6 ribonucleolytic action could also be demonstrated in vitro toward RNAs whose expression increased in the VapBC6-deficient strain during heat shock. These findings provide a biochemical mechanism and identify cellular targets underlying VapBC6-mediated control over microbial growth and survival at temperature extremes.

Yeast Colony Survival Depends on Metabolic Adaptation and Cell Differentiation Rather Than on Stress Defense

Enzymes scavenging reactive oxygen species (ROS) are important for cell protection during stress and aging. A deficiency in these enzymes leads to ROS imbalance, causing various disorders in many organisms, including yeast. In contrast to liquid cultures, where fitness of the yeast population depends on its ROS scavenging capability, the present study suggests that Saccharomyces cerevisiae cells growing in colonies capable of ammonia signaling use a broader protective strategy. Instead of maintaining high levels of antioxidant enzymes for ROS detoxification, colonies activate an alternative metabolism that prevents ROS production. Colonies of the strain deficient in cytosolic superoxide dismutase Sod1p thus developed the same way as wild type colonies. They produced comparable levels of ammonia and underwent similar developmental changes (expression of genes of alternative metabolism and center margin differentiation in ROS production, cell death occurrence, and activities of stress defense enzymes) and did not accumulate stress-resistant suppressants. An absence of cytosolic catalase Ctt1p, however, brought colonies developmental problems, which were even more prominent in the absence of mitochondrial Sod2p. sod2Delta and ctt1Delta colonies failed in ammonia production and sufficient activation of the alternative metabolism and were incapable of center margin differentiation, but they did not increase ROS levels. These new data indicate that colony disorders are not accompanied by ROS burst but could be a consequence of metabolic defects, which, however, could be elicited by imbalance in ROS produced in early developmental phases. Sod2p and homeostasis of ROS may participate in regulatory events leading to ammonia signaling.

Proteomic Analysis of a Nutritional Shift-up in Saccharomyces cerevisiae Identifies Gvp36 as a BAR-containing Protein Involved in Vesicular Traffic and Nutritional Adaptation

Yeast cells undergoing a nutritional shift-up from a poor to a rich carbon source take several hours to adapt to the novel, richer carbon source. The budding index is a physiologically relevant "global" parameter that reflects the complex links between cell growth and division that are both coordinately and deeply affected by nutritional conditions. We used changes in budding index as a guide to choose appropriate, relevant time points during an ethanol to glucose nutritional shift-up for preparation of samples for the analysis of proteome by two-dimensional electrophoresis/mass spectrometry. About 600 spots were detected. 90 spots, mostly comprising proteins involved in intermediary metabolism, protein synthesis, and response to stress, showed differential expression after glucose addition. Among modulated proteins we identified a protein of previously unknown function, Gvp36, showing a transitory increase corresponding to the drop of the fraction of budded cells. A gvp36Delta strain shares several phenotypes (including general growth defects, heat shock, and high salt sensitivity, defects in polarization of the actin cytoskeleton, in endocytosis and in vacuolar biogenesis, defects in entering stationary phase upon nutrient starvation) with secretory pathway mutants and with mutants in genes encoding the two previously known yeast BAR proteins (RSV161 and RSV167). We thus propose that Gvp36 represents a novel yeast BAR protein involved in vesicular traffic and in nutritional adaptation.

Farnesol and dodecanol effects on the Candida albicans Ras1‐cAMP signalling pathway and the regulation of morphogenesis

Candida albicans hypha formation which has been stimulated via the Ras1-cAMP-Efg1 signalling cascade is inhibited by farnesol, a C. albicans autoregulatory factor, and small molecules such as dodecanol. In cultures containing farnesol or dodecanol, hypha formation was restored upon addition of dibutyryl-cAMP. The CAI4-Ras1(G13V) strain, which carries a dominant-active variant of Ras1 and forms hyphae in the absence of inducing stimuli, grew as yeast in medium with farnesol or dodecanol; the heat shock sensitivity of the CAI4-Ras1(G13V) strain was also suppressed by these compounds. Neither Pde1 nor Pde2 was necessary for the repression of hyphal growth by farnesol or dodecanol. Two transcripts, CTA1 and HSP12, which are at higher levels upon mutation of Ras1 or Cdc35, were increased in abundance in cells grown with farnesol or dodecanol. Microscopic analysis of strains carrying CTA1 and HWP1 promoter fusions grown with intermediate concentrations of farnesol or dodecanol indicated a link between cells with the increased expression of cAMP-repressed genes and cells repressed for hypha formation. Because several cAMP-controlled outputs are affected by farnesol and dodecanol, our findings suggest that these compounds impact activity of the Ras1-Cdc35 pathway, thus leading to an alteration of C. albicans morphology.

Mutual Interdependence of MSI1 ( CAC3) and YAK1 in Saccharomyces cerevisiae

The MSI1 ( CAC3) gene of Saccharomyces cerevisiae has been implicated in diverse cellular functions, including suppression of the RAS/cAMP/protein kinase A signaling pathway, chromatin assembly and transcriptional co-repression. Seeking to identify the molecular mechanisms by which Msi1p carries out these distinct activities, a novel genetic interaction was uncovered with YAK1, which encodes a kinase that antagonizes the RAS/cAMP pathway. MSI1 was capable of efficiently suppressing the heat shock sensitivity caused by deletion of yak1. Surprisingly, the YAK1 gene is required for Msi1p to associate with Cac1p in the yeast two-hybrid system. A new activity of Msi1p was identified: the ability to activate transcription of a reporter gene when tethered near the promoter, but only in the absence of fermentable carbon sources. This transcriptional activation function was diminished substantially by the loss of YAK1. Furthermore, MSI1 influences YAK1 function; over-expression of YAK1 decreased the growth rate, but only in the presence of a functional MSI1 gene. Finally, it is shown that YAK1 antagonizes nuclear accumulation of Msi1p in non-fermenting cells. Taken together, these data demonstrate a novel interaction between Msi1p and Yak1p in which each protein influences the activity of the other.

The N-terminal region of the Saccharomyces cerevisiae RasGEF Cdc25 is required for nutrient-dependent cell-size regulation

In the yeast Saccharomyces cerevisiae, the Cdc25/Ras/cAMP/protein kinase A (PKA) pathway plays a major role in the control of metabolism, stress resistance and proliferation, in relation to the available nutrients and conditions. The budding yeast RasGEF Cdc25 was the first RasGEF to be identified in any organism, but very little is known about its activity regulation. Recently, it was suggested that the dispensable N-terminal domain of Cdc25 could negatively control the catalytic activity of the protein. In order to investigate the role of this domain, strains were constructed that produced two different versions of the C-terminal domain of Cdc25 (aa 907-1589 and 1147-1589). The carbon-source-dependent cell size control mechanism present in the wild type was found in the first of these mutants, but was lost in the second mutant, for which the cell size, determined as protein content, was the same during exponential growth in both ethanol- and glucose-containing media. A biparametric analysis demonstrated that this effect was essentially due to the inability of the mutant producing the shorter sequence to modify its protein content at budding. A similar phenotype was observed in strains that lacked CDC25, but which possessed a mammalian GEF catalytic domain. Taken together, these results suggest that Cdc25 is involved in the regulation of cell size in the presence of different carbon sources. Moreover, production of the aa 876-1100 fragment increased heat-stress resistance in the wild-type strain, and rescued heat-shock sensitivity in the ira1Delta background. Further work will aim to clarify the role of this region in Cdc25 activity and Ras/cAMP pathway regulation.

Residues Distant from the Active Site Influence Protein-tyrosine Phosphatase 1B Inhibitor Binding

Regions of protein-tyrosine phosphatase (PTP) 1B that are distant from the active site yet affect inhibitor binding were identified by a novel library screen. This screen was based on the observation that expression of v-Src in yeast leads to lethality, which can be rescued by the coexpression of PTP1B. However, this rescue is lost when yeast are grown in the presence of PTP1B inhibitors. To identify regions of PTP1B (amino acids 1-400, catalytic domain plus 80-amino acid C-terminal tail) that can affect the binding of the difluoromethyl phosphonate (DFMP) inhibitor 7-bromo-6-difluoromethylphosphonate 3-naphthalenenitrile, a library coexpressing PTP1B mutants and v-Src was generated, and the ability of yeast to grow in the presence of the inhibitor was evaluated. PTP1B inhibitor-resistant mutations were found to concentrate on helix alpha7 and its surrounding region, but not in the active site. No resistant amino acid substitutions were found to occur in the C-terminal tail, suggesting that this region has little effect on active-site inhibitor binding. An in-depth characterization of a resistant substitution localizing to region alpha7 (S295F) revealed that this change minimally affected enzyme catalytic activity, but significantly reduced the potency of a panel of structurally diverse DFMP PTP1B inhibitors. This loss of inhibitor potency was found to be due to the difluoro moiety of these inhibitors because only the difluoro inhibitors were shifted. For example, the inhibitor potency of a monofluorinated or non-fluorinated analog of one of these DFMP inhibitors was only minimally affected. Using this type of library screen, which can scan the nearly full-length PTP1B sequence (catalytic domain and C-terminal tail) for effects on inhibitor binding, we have been able to identify novel regions of PTP1B that specifically affect the binding of DFMP inhibitors.

Increased ubiquitin-dependent degradation can replace the essential requirement for heat shock protein induction.

Serine palmitoyltransferase, the first enzyme in ceramide biosynthesis, is required for resistance to heat shock. We show that increased heat shock sensitivity in the absence of serine palmitoyltransferase activity correlates with a lack of induction of the major heat shock proteins (Hsps) at high temperature. Normal heat shock resistance can be restored, without restoration of ceramide synthesis or induction of Hsps, by overexpression of ubiquitin. This function of ubiquitin requires the proteasome. These data imply that the essential function of Hsp induction is the removal of misfolded or aggregated proteins, not their refolding. This suggests that cells stressed by heat shock do not die because of the loss of protein activity due to their denaturation, but because of the inherent toxicity of the denatured and/or aggregated proteins.

Inhibition of an activated Ras protein with genetically selected peptide aptamers.

Mutant alleles of Ras maintain an activated, GTP-bound conformation and relay mitogenic signals that cannot be turned off. A genetic selection in Saccharomyces cerevisiae was used to identify peptide aptamers that suppress the growth arrest phenotype of an activated Ras allele. Peptide aptamers were expressed as C-terminal fusions to glutathione-S-transferase. Modifications that alter the coding capacity of the peptide aptamer indicate it is necessary for Ras2-Val19 suppression. Aptamer expression also reduces the elevated levels of cAMP and suppresses the heat shock sensitivity characteristic of Ras-activated yeast cells. The peptide aptamer retains suppressor activity when fused to thioredoxin. The peptide aptamer expression strategy described here indicates that aptamers presented as unconstrained peptides have functional capacity in vivo.

Krh1p and Krh2p act downstream of the Gpa2p G(alpha) subunit to negatively regulate haploid invasive growth.

The yeast G(alpha) subunit Gpa2p and its coupled receptor Gpr1p function in a signaling pathway that is required for the transition to pseudohyphal and invasive growth. A two-hybrid screen using a constitutively active allele of GPA2 identified the KRH1 gene as encoding a potential binding partner of Gpa2p. Strains containing deletions of KRH1 and its homolog KRH2 were hyper-invasive and displayed a high level of expression of FLO11, a gene involved in pseudohyphal and invasive growth. Therefore, KRH1 and KRH2 encode negative regulators of the invasive growth pathway. Cells containing krh1Delta krh2Delta mutations also displayed increased sensitivity to heat shock and decreased sporulation efficiency, indicating that Krh1p and Krh2p regulate multiple processes controlled by the cAMP/PKA pathway. The krh1Delta krh2Delta mutations suppressed the effect of a gpa2Delta mutation on FLO11 expression and eliminated the effect of a constitutively active GPA2 allele on induction of FLO11 and heat shock sensitivity, suggesting that Krh1p and Krh2p act downstream of Gpa2p. The Sch9p kinase was not required for the signal generated by deletion of KRH1 and KRH2; however, the cAMP-dependent kinase Tpk2p was required for generation of this signal. These results support a model in which activation of Gpa2p relieves the inhibition exerted by Krh1p and Krh2p on components of the cAMP/PKA signaling pathway.

Critical Function of the Ras-associating Domain as a Primary Ras-binding Site for Regulation of Saccharomyces cerevisiaeAdenylyl Cyclase

Mammalian candidate effectors of the small GTPase Ras, such as RalGDS, afadin/AF-6, Rin1, and phospholipase Cepsilon, have been shown to share structurally conserved modules termed Ras-associating (RA) domains at their Ras-binding sites. The Ras-binding domains of Raf-1 and phosphoinositide 3-kinase gamma (other Ras effectors) also share a similar tertiary structure with the RA domains. On the other hand, the primary Ras-binding site of Saccharomyces cerevisiae adenylyl cyclase, the best characterized Ras effector, has been mapped by mutational studies to the leucine-rich repeats (LRR) domain (amino acids 674-1300), whose structure apparently bears no resemblance to the RA domains. By a computer algorithm-based search we have unexpectedly found an RA domain in the N-terminal 81 amino acid residues (676) of the LRR domain. The purified RA-domain polypeptide exhibits an ability to bind directly to Ras in a GTP-dependent manner and to competitively inhibit Ras-dependent activation of adenylyl cyclase in vitro, with an affinity comparable with that of the whole LRR domain. The specificity of binding of the RA domain to various Ras effector region mutants is indistinguishable from that of the full-length adenylyl cyclase. The activated RAS2 (RAS2(Val-19))-dependent heat shock sensitivity of yeast cells is suppressed by overexpression of the RA domain polypeptide. Further, mutations of the RA domain abolish its Ras binding activity, and adenylyl cyclase molecules carrying these mutations are rendered unactivatable by Ras in vitro. This RA domain bears highest similarity to the Ras-binding domain of Raf-1 based on comparison of its primary and predicted secondary structures with those of other Ras effectors. These results indicate that the RA domain is a primary Ras-binding site for activation of adenylyl cyclase, implicating RA domains as universal modules for interaction of effectors with Ras, conserved from yeast to mammals.

Yeast Rpi1 is a putative transcriptional regulator that contributes to preparation for stationary phase.

The RPI1 gene of Saccharomyces cerevisiae was identified initially as a dosage suppressor of the heat shock sensitivity associated with overexpression of RAS2 (J. Kim and S. Powers, Mol. Cell. Biol. 11:3894-3904, 1991). Based on its failure to suppress mutationally activated RAS2, RPII was proposed to be a negative regulator of the Ras/cyclic AMP (cAMP) pathway that functions at a point upstream of Ras. We isolated RPI1 as a high-copy-number suppressor of the cell lysis defect associated with a null mutation in the MPK1 gene, which encodes the mitogen-activated protein kinase of the cell wall integrity-signaling pathway. Although the sequence of Rpil is not informative about its function, we present evidence that this protein resides in the nucleus, possesses a transcriptional activation domain, and affects the mRNA levels of several cell wall metabolism genes. In contrast to the previous report, we found that RPI1 overexpression suppresses defects associated with mutational hyperactivation of the Ras/cAMP pathway at all points including constitutive mutations in the cAMP-dependent protein kinase. We present additional genetic and biochemical evidence that Rpil functions independently of and in opposition to the Ras/cAMP pathway to promote preparations for the stationary phase. Among these preparations is a fortification of the cell wall that is antagonized by Ras pathway activity. This observation reveals a novel link between the Ras/cAMP pathway and cell wall integrity. Finally, we propose that inappropriate expression of RPI1 during log phase growth drives fortification of the cell wall and that this behavior is responsible for suppression of the mpkl cell lysis defect.